CN103266106A - Total DNA (Deoxyribonucleic Acid) extraction method of bottom mud containing high content of humus at river mouth - Google Patents
Total DNA (Deoxyribonucleic Acid) extraction method of bottom mud containing high content of humus at river mouth Download PDFInfo
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Abstract
The invention discloses a total DNA (Deoxyribonucleic Acid) extraction method of bottom mud containing high content of humus at the river mouth. The method comprises the following steps: washing bottom mud by stroke-physiological saline solution to remove part of impurities such humus, and meanwhile, concentrating microorganisms; then, multigelating in liquid nitrogen and a water bath at 65 DEG C by means of CTAB (Cetyltrimethyl Ammonium Bromide), proteinase K and lysozyme for cell disruption, and then adding SDS (Sodium Dodecyl Sulfonate) and insulating for 2 hours in the water bath at 65 DEG C to combine protein; adding isovolumetric phenol/chloroform/isoamylol (25:24:1) to remove protein; adding isovolumetric isopropanol to set DNA, washing precipitate with 70% ethanol, and dissolving DNA by TE (pH8.0) after airing; adding 3 times of absolute ethyl alcohol in volume to re-set DNA, washing the precipitate with 70% ethanol, dissolving the precipitated DNA by TE after airing, and storing at minus 20 DEG C for later use. The method provided by the invention is simple to operate and cheap in price. The total DNA of bottom mud extracted by the method provided by the invention has the advantages of high purity and integral biodiversity, and can be used for molecular biology study and microbial diversity analysis.
Description
Technical field
The invention belongs to environmental microbiology and technical field of molecular biology, specifically set forth a kind of high high efficiency method that contains soil ulmin river mouth bed mud total DNA extraction.
Background technology
The river mouth bed mud is the same with top soil, all contain abundant microorganism, but these microorganism overwhelming majority also can not cultivate in the laboratory, thereby the work that can't conduct a research, and the development of grand genomics has in recent years brought hope for the research of soil microorganisms.Extracting method about soil microbial DNA has many reports in recent years, but few at the relevant report of the effective extracting method of ditch bed mud microbial DNA.The a large amount of soil ulmin that contain in the soil are to cause the soil microorganisms high quality DNA to extract the major cause of difficulty, yet the impurity such as soil ulmin that contain in the ditch bed mud are more complicated, so the extraction difficulty of ditch bed mud microorganism high quality DNA is bigger.Usually contain impurity such as a large amount of soil ulmin, pigment among the bottom mud microbe DNA that extracts, can not carry out follow-up molecular biology research, for removing these impurity, mainly contain two kinds of ways: a kind of way is before extracting DNA the bed mud sample to be handled, and this method is little to the high bed mud sample effect of humus content; Another kind of way is that this way all is effectively to most of bed mud samples with DNA specific adsorption column purification bottom mud microbe DNA, but price is relatively costly.In addition, have multiple soil DNA to extract test kit on the market, these test kits are not only expensive, and also bad to the extraction effect of the high ditch bed mud of humus content or soil DNA.
Summary of the invention
The object of the present invention is to provide a kind ofly to contain the soil ulmin river mouth bed mud method of extracting high quality DNA from height, present method is easy to operate, and is cheap, overcome existing method and extracted the high total DNA of the soil ulmin river mouth bed mud defective of low quality that contains.
The present invention contains the method for extracting high quality DNA the soil ulmin river mouth bed mud from height, may further comprise the steps:
1. bed mud The pretreatment: take by weighing 10g river mouth bed mud and put into the 50ml centrifuge tube, add 30ml physiological saline, vortex vibration 5min, leave standstill 2min, first 2500r/min low-speed centrifugal 3min under 4 ℃, 10000r/min high speed centrifugation 8min abandons supernatant again, gets the top 1/2 of precipitation and transfers in another new centrifuge tube; Repeat to go on foot 2 times, will obtain the 1.25g bed mud and transfer in the 10ml centrifuge tube; Wash bed mud with physiological saline, at 4 ℃ of centrifugal 8min of following 10000r/min, till the supernatant of washing times after centrifugal is water white transparency.
2. the lysis of bottom mud microbe: 1.25g adds 2.5mL CTAB solution, the abundant mixing of vortex through pretreated bed mud; Multigelation is 3 times in liquid nitrogen and 65 ℃ of water-baths; Under 37 ℃, shake 30min.
3.DNA separation: add 10% SDS to 2%, 65 ℃ of water bath heat preservation 2h of final concentration, put upside down mixing once every 15-20min; The centrifugal 10min of room temperature 12000r/min collects supernatant; On reset and add equal-volume phenol/chloroform/primary isoamyl alcohol (25: 24: 1), put upside down mixing gently; The centrifugal 10min of room temperature 12000r/min stays supernatant; Phenol/chloroform/primary isoamyl alcohol (25: 24: 1) extracting 3 times; On reset and add isopyknic Virahol, the precipitation 1-2h; Under 4 ℃, with the centrifugal 10min of 12000r/min, abandon supernatant; Freezing washing with alcohol DNA precipitation with 70% under 4 ℃, with the centrifugal 5min of 12000r/min, is stayed precipitation; 70% freezing washing with alcohol DNA precipitation 3 times, seasoning; With TE (pH8.0) dissolving DNA precipitation, obtain the total DNA crude extract of river mouth bed mud.
4.DNA purifying: the DNA crude extract adds the freezing dehydrated alcohol of 3 times of volumes in the step 3, at-20 ℃ of following precipitation 1-2h; Under 4 ℃, with the centrifugal 10min of 12000r/min, stay precipitation; Precipitation under 4 ℃, with the centrifugal 5min of 12000r/min, is stayed precipitation, seasoning with 70% freezing washing with alcohol 2 times; With TE (pH8.0) dissolving DNA precipitation, obtain the total DNA of final river mouth bed mud.DNA runs 0.8% agarose gel electrophoresis.
5.16S the PCR of rDNA: the DNA that extracts with step 4 is template, carry out PCR with bacterial 16 S rDNA universal primer 63F (5 '-CAGGCCTAACACATGCAAGTC-3 ') and 1387R (5 '-GGGCGGWGTGTACAAGGC-3 '), the PCR product runs 1% agarose gel electrophoresis.
Physiological saline is 0.85% NaCl solution in the described step 1, is adjusted to pH=9.5 with 4M NaOH solution, 121 ℃ of autoclaving 20min; CTAB solution in the described step 2 is the Tris-HCl of 100mM by final concentration, the EDTA of 100mM, and the NaCl of 1.5M, 1% CTAB, the N,O-Diacetylmuramidase of 1mg/ml and the Proteinase K of 0.2mg/ml are formed (N,O-Diacetylmuramidase and Proteinase K time spent add again).
The advantage that the present invention has:
1. the present invention improves the extracting method of the total DNA of river mouth bed mud, has improved the quality of the total DNA of bed mud.The total DNA electrophoretogram that extracts in the bed mud sample is a line, illustrates that DNA purity height, impurity are few, become follow-up molecular biology experiment and are more prone to.
2. river mouth of the present invention bed mud total DNA extraction method has wide range of applications, be not only applicable to various height and contain the extraction of the total DNA of ditch bed mud of soil ulmin, be applicable to that also various height contain the extraction of the total DNA of soil of soil ulmin, more can be used for containing the extraction of the few ditch bed mud of soil ulmin and the total DNA of soil.
3. river mouth of the present invention bed mud total DNA extraction method can keep bottom mud microbe various, makes the various analysis of bottom mud microbe genuine and believable.
4. river mouth of the present invention bed mud total DNA extraction method is simple, cheap to the DNA purification process.
Description of drawings
Fig. 1 is 0.8% agarose gel electrophoretogram of river mouth bed mud and the total DNA of soil.Be numbered total DNA that 1,2,3 swimming lane is respectively Chang River river mouth bed mud, river mouth, Ganjiang River bed mud and Meiling Hill soil, M1 is λ-Hind III digest DNA Marker, and M2 is 1kb DNA Marker.
Fig. 2 is 1% agarose gel electrophoretogram of the 16S rDNA pcr amplification product of river mouth bed mud and the total DNA of soil.Be numbered the 16S rDNA pcr amplification result that 1,2,3 swimming lane is respectively Chang River river mouth bed mud, river mouth, Ganjiang River bed mud and the total DNA of Meiling Hill soil, M3 is DL2000DNA Marker.
Embodiment
Below in conjunction with specific embodiment the present invention is described in further detail.
Utilize the method for the invention to extract the total DNA of Chang River river mouth bed mud.Chang River river mouth bed mud is got the bed mud of upper strata 0-3cm as sample from the west side, 200m place, upstream of Chang River and river, Lean intersection, and this bed mud sample is grey black, the humus content height.Below be concrete implementation step:
1. bed mud The pretreatment: take by weighing 10g river mouth bed mud and put into the 50ml centrifuge tube, add 30ml physiological saline, vortex vibration 5min, leave standstill 2min, first 2500r/min low-speed centrifugal 3min under 4 ℃, 10000r/min high speed centrifugation 8min abandons supernatant again, gets the top 1/2 of precipitation and transfers in another new centrifuge tube; Repeat to go on foot 2 times, will obtain the 1.25g bed mud and transfer in the 10ml centrifuge tube; Wash bed mud with physiological saline, at 4 ℃ of centrifugal 8min of following 10000r/min, till the supernatant of washing times after centrifugal is water white transparency.
2. the lysis of bottom mud microbe: 1.25g adds 2.5mL CTAB solution, the abundant mixing of vortex through pretreated bed mud; Multigelation is 3 times in liquid nitrogen and 65 ℃ of water-baths; Under 37 ℃, shake 30min.
3.DNA separation: add 10% SDS to 2%, 65 ℃ of water bath heat preservation 2h of final concentration, put upside down mixing once every 15-20min; The centrifugal 10min of room temperature 12000r/min collects supernatant; On reset and add equal-volume phenol/chloroform/primary isoamyl alcohol (25: 24: 1), put upside down mixing gently; The centrifugal 10min of room temperature 12000r/min stays supernatant; Phenol/chloroform/primary isoamyl alcohol (25: 24: 1) extracting 3 times; On reset and add isopyknic Virahol, the precipitation 1-2h; Under 4 ℃, with the centrifugal 10min of 12000r/min, abandon supernatant; Freezing washing with alcohol DNA precipitation with 70% under 4 ℃, with the centrifugal 5min of 12000r/min, is stayed precipitation; 70% freezing washing with alcohol DNA precipitation 3 times, seasoning; With TE (pH8.0) dissolving DNA precipitation, obtain the total DNA crude extract of river mouth bed mud.
4.DNA purifying: the DNA crude extract adds the freezing dehydrated alcohol of 3 times of volumes in the step 3, at-20 ℃ of following precipitation 1-2h; Under 4 ℃, with the centrifugal 10min of 12000r/min, stay precipitation; Precipitation under 4 ℃, with the centrifugal 5min of 12000r/min, is stayed precipitation, seasoning with 70% freezing washing with alcohol 2 times; With TE (pH8.0) dissolving DNA precipitation, obtain the total DNA of final river mouth bed mud.DNA runs 0.8% agarose gel electrophoresis.
5.16S the PCR of rDNA: the DNA that extracts with step 4 is template, carry out PCR with bacterial 16 S rDNA universal primer 63F (5 '-CAGGCCTAACACATGCAAGTC-3 ') and 1387R (5 '-GGGCGGWGTGTACAAGGC-3 '), the PCR product runs 1% agarose gel electrophoresis.The PCR parameter is 95 ℃ of sex change 5min; 95 ℃ of sex change 35s, 54 ℃ of annealing 40s, 72 ℃ are extended 100s, 30 circulations; 72 ℃ are extended 8min.
Physiological saline is 0.85% NaCl solution in the described step 1, is adjusted to pH=9.5 with 4M NaOH solution, 121 ℃ of autoclaving 20min; CTAB solution in the described step 2 is the Tris-HCl of 100mM by final concentration, the EDTA of 100mM, and the NaCl of 1.5M, 1% CTAB, the N,O-Diacetylmuramidase of 1mg/ml and the Proteinase K of 0.2mg/ml are formed (N,O-Diacetylmuramidase and Proteinase K time spent add again).
As shown in Figure 1, swimming lane 1DNA electrophoretogram band is single clear, and no conditions of streaking illustrates DNA purity height, not fracture; As shown in Figure 2, the PCR product electrophoretogram of swimming lane 1 can be seen the purpose segment of about 1380bp clearly, and the total DNA quality height that utilizes the method for the invention to extract Chang River river mouth bed mud is described, can be used for follow-up molecular biosciences research.
Utilize the method for the invention to extract the total DNA of river mouth, Ganjiang River bed mud.River mouth, Ganjiang River bed mud is got the bed mud of upper strata 0-3cm as sample from Ganjiang River and the north side, 150m place, upstream of repairing river intersection, and this bed mud sample is yellow clay, contains soil ulmin, particularly the pigment content height.
Concrete implementation step is identical with embodiment 1.
As shown in Figure 1, swimming lane 2DNA electrophoretogram band is single clear, and no conditions of streaking illustrates DNA purity height, not fracture; As shown in Figure 2, the PCR product electrophoretogram of swimming lane 2 can be seen the purpose segment of about 1380bp clearly, and the total DNA quality height that utilizes the method for the invention to extract river mouth, Ganjiang River bed mud is described, can be used for follow-up molecular biosciences research.
Utilize the method for the invention to extract the total DNA of soil.Soil is got the corrupt many topsoils of falling leaves on the mountain as sample from Meiling Hill, Nanchang City, Jiangxi Province, and this pedotheque is grey black, contains a large amount of soil ulmin.
Concrete implementation step is identical with embodiment 1.
As shown in Figure 1, swimming lane 3DNA electrophoretogram band is single clear, and no conditions of streaking illustrates DNA purity height, not fracture; As shown in Figure 2, the PCR product electrophoretogram of swimming lane 3 can be seen the purpose segment of about 1380bp clearly, and the total DNA quality height that utilizes the method for the invention to extract Meiling Hill soil is described, can be used for follow-up molecular biosciences research.
Claims (4)
1. one kind contains the method for extracting total DNA the river mouth bed mud of soil ulmin from height, it is characterized in that, may further comprise the steps:
(1) bed mud The pretreatment:
1. take by weighing 10g river mouth bed mud and put into the 50ml centrifuge tube, add 30ml physiological saline, vortex vibration 5min, leave standstill 2min, first 2500r/min low-speed centrifugal 3min, 10000r/min high speed centrifugation 8min again under 4 ℃, abandon supernatant, get the top 1/2 of precipitation and transfer in another new centrifuge tube;
2. repeat to go on foot 2 times, will obtain the 1.25g bed mud and transfer in the 10ml centrifuge tube;
3. wash bed mud with physiological saline, at 4 ℃ of centrifugal 8min of following 10000r/min, till the supernatant of washing times after centrifugal is water white transparency;
(2) lysis of bottom mud microbe:
1. 1.25g adds 2.5mL CTAB solution, the abundant mixing of vortex through pretreated bed mud;
2. multigelation 3 times in liquid nitrogen and 65 ℃ of water-baths;
3. under 37 ℃, shake 30min;
(3) separation of DNA:
1. add 10% SDS to 2%, 65 ℃ of water bath heat preservation 2h of final concentration, put upside down mixing once every 15-20min;
2. the centrifugal 10min of room temperature 12000r/min collects supernatant;
3. reset and add equal-volume phenol/chloroform/primary isoamyl alcohol (25: 24: 1) on, put upside down mixing gently; The centrifugal 10min of room temperature 12000r/min stays supernatant; Phenol/chloroform/primary isoamyl alcohol (25: 24: 1) extracting 3 times;
4. reset and add isopyknic Virahol on, precipitation 1-2h; Under 4 ℃, with the centrifugal 10min of 12000r/min, abandon supernatant;
5. with 70% freezing washing with alcohol DNA precipitation, under 4 ℃, with the centrifugal 5min of 12000r/min, stay precipitation; 70% freezing washing with alcohol DNA precipitation 3 times, seasoning;
6. use TE (pH8.0) dissolving DNA precipitation, obtain the total DNA crude extract of river mouth bed mud;
(4) purifying of DNA:
1. the middle DNA crude extract of step (3) adds the freezing dehydrated alcohol of 3 times of volumes, precipitates 1-2h down at-20 ℃; Under 4 ℃, with the centrifugal 10min of 12000r/min, stay precipitation;
2. precipitation under 4 ℃, with the centrifugal 5min of 12000r/min, is stayed precipitation, seasoning with 70% freezing washing with alcohol 2 times;
3. use TE (pH8.0) dissolving DNA precipitation, obtain the total DNA of final river mouth bed mud; DNA runs 0.8% agarose gel electrophoresis;
(5) PCR of 16S rDNA: the DNA that extracts with step (4) is template, carry out PCR with bacterial 16 S rDNA universal primer 63F (5 '-CAGGCCTAACACATGCAAGTC-3 ') and 1387R (5 '-GGGCGGWGTGTACAAGGC-3 '), the PCR product runs 1% agarose gel electrophoresis.
2. the method for claim 1, it is characterized in that the physiological saline of described step (1): 0.85% NaCl solution is adjusted to pH=9.5 with 4M NaOH solution, 121 ℃ of autoclaving 20min.
3. the method for claim 1, the CTAB solution that it is characterized in that described step (2), be the Tris-HCl of 100mM by final concentration, the EDTA of 100mM, 1.5M NaCl, 1% CTAB, the N,O-Diacetylmuramidase of 1mg/ml and the Proteinase K of 0.2mg/ml are formed (N,O-Diacetylmuramidase and Proteinase K time spent add again).
4. the application of the method for claim 1 in various types of ditch bed muds and soil total DNA extraction.
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| CN104560951A (en) * | 2014-12-03 | 2015-04-29 | 复旦大学泰州健康科学研究院 | Extraction method of metagenome DNA and kit for extraction method |
| CN106047869A (en) * | 2016-08-23 | 2016-10-26 | 厦门基源医疗科技有限公司 | Extraction method of metagenome of microbes in seawater |
| CN107142258A (en) * | 2017-06-21 | 2017-09-08 | 长沙金域医学检验所有限公司 | Stickiness sample DNA extracting method in HPV detections |
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| CN112501156A (en) * | 2020-11-20 | 2021-03-16 | 中国水产科学研究院黄海水产研究所 | High-efficiency extraction method of total DNA of marine shellfish biological sediment |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104087573A (en) * | 2014-06-18 | 2014-10-08 | 福建农林大学 | Extraction method for microbial genome DNA in water culture solution |
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| CN106047869A (en) * | 2016-08-23 | 2016-10-26 | 厦门基源医疗科技有限公司 | Extraction method of metagenome of microbes in seawater |
| CN106047869B (en) * | 2016-08-23 | 2019-05-24 | 厦门基源医疗科技有限公司 | A kind of extracting method of the macro genome of seawater microorganism |
| CN107142258A (en) * | 2017-06-21 | 2017-09-08 | 长沙金域医学检验所有限公司 | Stickiness sample DNA extracting method in HPV detections |
| CN107475249A (en) * | 2017-09-11 | 2017-12-15 | 广东美格基因科技有限公司 | Method that is a kind of low from biomass and being rich in extraction microorganism total DNA in the soil of humus |
| CN112501156A (en) * | 2020-11-20 | 2021-03-16 | 中国水产科学研究院黄海水产研究所 | High-efficiency extraction method of total DNA of marine shellfish biological sediment |
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Application publication date: 20130828 |